1. Field of the Invention
The present invention relates to a process for the enzymatic production of D-pantothenic acid and/or its salts or mixtures containing the same, using microorganisms of the family Enterobacteriaceae in which at least the serC gene is enhanced.
2. Description of the Background
Several thousands of tons of pantothenic are produced worldwide each year. Pantothenic acid is used among other things in human medicine, in the pharmaceutical industry and in the food industry. A large part of the pantothenic acid that is produced is used as an animal feedstuff for commercially important animals such as poultry and pigs. The worldwide demand for pantothenic acid is rising.
Pantothenic acid may be produced by chemical synthesis or biotechnologically by fermenting suitable microorganisms in suitable nutrient solutions. In the chemical synthesis DL-pantolactone is an important precursor. DL-pantolactone is produced in a multi-stage process from formaldehyde, isobutyl aldehyde and cyanide, in which in further process steps the racemic mixture is separated and the D-panto-lactone is condensed with xcex2-alanine to produce the desired D-pantothenic acid.
The typical commercial form is the calcium salt of D-pantothenic acid. The calcium salt of the racemic mixture of D,L-pantothenic acid is also commonly used.
The advantage of the enzymatic production by microorganisms is the direct formation of the desired stereoisomer form, namely the D-form, which is free of L-pantothenic acid.
Various species of bacteria, such as for example Escherichia coli (E. coli), Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacterium ammoniagenes and also yeasts, such as for example Debaromyces castellii, can produce D-pantothenic acid in a nutrient solution containing glucose, DL-pantoic acid and xcex2-alanine, as demonstrated in EP-A 0 493 060. EP-A 0 493 060 furthermore shows that with E. coli the formation of D-pantothenic acid in a nutrient solution containing glucose, DL-pantoic acid and xcex2-alanine is improved by amplification of pantothenic acid biosynthesis genes from E. coli that are contained on the plasmids pFV3 and pFV5.
EP-A 0 590 857 and U.S. Pat. No. 5,518,906 describe mutants derived from E. coli strain IF03547, such as FV5714, FV525, FV814, FV521, FV221, FV6051 and FV5069, that exhibit resistance to various antimetabolites such as salicylic acid, a-ketobutyric acid, xcex2-hydroxyaspartic acid, O-methyl-threonine and xcex1-ketoisovaleric acid. These mutants produce pantoic acid in a nutrient solution containing glucose, and produce D-pantothenic acid in a nutrient solution containing glucose and xcex2-alanine. EP-A 0 590 857 and U.S. Pat. No. 5,518,906 also show that, after amplification of the pantothenic acid biosynthesis genes panB, panC and panD that are said to be contained on the plasmid pFV31, in the aforementioned strains the production of D-pantoic acid is improved in glucose-containing nutrient solutions, while the production of D-pantothenic acid is improved in a nutrient solution that contains glucose and xcex2-alanine.
Furthermore, the promoting action of the enhancement of the ilvGM operon on the production of D-pantothenic acid is reported in WO 97/10340. Finally, the effect of the enhancement of the panE gene on the formation of D-pantothenic acid is reported in EP-A-1001027.
According to known methos, D-pantothenic acid or the corresponding salt is isolated from the fermentation broth and purified (EP-A-0590857 and WO 96/33283) and consequently used in purified form, or the fermentation broth containing D-pantothenic acid is dried as a whole (EP-A-1050219) and used in particular as a feedstuffs additive.
In view of the increasing demand for D-pantothenic acid, there remains a need for new methods for producing this material.
It is an object of the present invention to provide new methods for the improved enzymatic production of D-pantothenic acid and/or its salts and/or feedstuffs additives containing the same.
The invention provides a method for the production of D-pantothenic acid and/or its salts or in addition to the latter the production of feedstuffs additives containing further constituents from the fermentation, by fermentation of microorganisms of the family Enterobacteriaceae, in particular those that already produce D-pantothenic acid, in which
a) the nucleotide sequence(s) in the microorganisms coding for the endogenous serC gene is amplified, in particular overexpressed, in conditions that are suitable for the production of 3-phosphoserine aminotransferase,
b) the D-pantothenic acid and/or its salts are enriched in the medium or in the cells of the microorganisms, and
c) the desired products are isolated after the end of the fermentation, the biomass and/or optionally further constituents of the fermentation broth being separated in an amount of 0 to 100%,
wherein the microorganisms produce D-pantothenic acid.
The invention also provides a process in which after the end of the fermentation the biomass remains partially or totally in the fermentation broth and the broth obtained in this way is worked up, optionally after concentration, to form a mixture containing solid D-pantothenic acid and/or its salts, which preferably contains further constituents of the fermentation broth.
Accordingly, the present invention provides a method of producing D-pantothenic acid and/or a salt thereof, comprising:
fermenting a microorganism of the family Enterobacteriaceae, in which the nucleotide sequence(s) in the microorganism coding for the endogenous serC gene is amplified, in a medium under conditions suitable for the production of 3-phosphoserine aminotransferase,
wherein the microorganism produces the D-pantothenic acid and/or a salt thereof.
The present invention also provides a method of producing a feedstuffs additive, comprising:
producing D-pantothenic acid and/or a salt thereof as described above, and
combining the D-pantothenic acid and/or a salt thereof with a carrier suitable for use in feedstuffs.
The present invention also provides a vector for the expression of the serC gene from E. coli, containing a promoter and the gene sequence.
The present invention also provides a microorganism of the family Enterobacteriaceae, transformed with the vector described above.
The present invention also provides a method of producing D-pantothenic acid and/or a salt thereof by fermentation of the microorganism described above.
The present invention also provides a method of producing a foodstuffs additive, comprising:
(a) producing D-pantothenic acid or a salt thereof as described above, wherein the alkaline earth metal of the alkaline earth salt is magnesium and/or calcium,
(a) optionally, removing water from the fermentation broth,
(b) separating the biomass formed during the fermentation is separated in an amount of 0 to 100%,
(c) optionally, adding one or more magnesium and/or calcium salts of D-pantothenic acid to the fermentation broths from (b), and
(d) producing the feedstuffs additive,
wherein the amount of the added one or more magnesium and/or calcium salts of D-pantothenic acid is such that the amount of in the feedstuffs additive is in the range from about 20 to 80 wt. % based on the dry mass of the feedstuffs additive.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following FIGURE in conjunction with the detailed description below.